The long-term objectives of the studies proposed here are to identify and characterize factors that play an important role in transcription initiation by RNA polymerase II in vivo. We will 1) investigate the role of TFIIA in the response to oxidative stress; 2) determine the function(s) of TFIID in transcriptional processes in vivo; and 3) further characterize the transcriptional requirement for the newly defined protein SPN1. To accomplish these aims, a combination of genetic, molecular and biochemical approaches will be employed in Saccharomyces cerevisiae. Specifically, we have isolated mutant alleles of TFIIA that are defective for response to the transcriptional activator YAP1, YAP1 is the key regulator of oxidative stress in yeast, and the response to oxidative stress is conserved from yeast to humans, In fact, oxidative stress appears to play a role in a variety of human conditions, including neurodegenerative diseases, cancer, and aging. Studies described in Specific Aim 1 involve a complete biochemical and genetic characterization of the TFIIA-YAP1 connection. These results will certainly advance our understanding of the mechanism of induction of the genes required for protection from oxidative damage. In studies described in Specific Aim 2, we will map the protein-protein interactions in the TFIID complex, and exploit this information to design mutants that specifically disrupt certain TAF-TAF interactions. We will also refine our knowledge about the TFIIA-TAF40 interaction, by isolating compensatory mutants. TAF mutants generated will be characterized at the genetic and biochemical level, with the goal of determining the essential nature of the protein-protein interactions specific for TFIID functions. Using a genetic selection for suppression of an activation defective allele of TBP, we have also identified a new gene involved in transcriptional processes: SPN1. To determine the mechanisms by which this essential and highly conserved protein functions in gene expression, studies in Specific Aim 3 are focused on further characterizing SPN1, and identifying proteins that interact with SPN1 via biochemical and genetic approaches. Results generated from this proposal should provide important information on the factors and mechanisms essential for transcriptional regulation directly relevant to the human condition, since the processes of transcriptional control are conserved from yeast to humans. [unreadable] [unreadable]